RSV, a member of the paramyxovirus family, is an enveloped RNA-containing virus that is the most important viral agent of pediatric respiratory tract disease worldwide. Accompanying reports describe the application of molecular techniques for the development of attenuated RSV vaccine viruses. The goal of the present report is to characterize the cis-acting sequence elements in the viral genome and to understand their role in encapsidation, RNA replication, transcription and virion assembly. This is of interest because RSV is relatively distinct from other paramyxoviruses, and thus information from model systems is of limited value. Also, a better knowledge of the organization and expression of the viral genome will help guide efforts to make attenuated recombinant viruses for use in vaccines. We previously described the development of cDNA-encoded helper-dependent minireplicon analogs of RSV genomic or antigenomic RNA which, when complemented by viral proteins supplied by coinfecting RSV or from plasmids, can program reconstituted genome encapsidation, transcription, RNA replication and virion morphogenesis. In the minireplicons, the authentic genomic termini are retained but the viral genes are deleted and replaced with one or more reporter genes, such as that for bacterial chloramphenicol acetyl transferase (CAT) or luciferase (LUC), each under the control of a set of the RSV sequence motifs which are thought to control transcription. Here, this system was used to identify and characterize the cis-acting replication and transcription signals in the RSV genome and to study the mechanism of gene expression.